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        INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

        WORLD HEALTH ORGANIZATION



        SAFETY EVALUATION OF CERTAIN
        FOOD ADDITIVES AND CONTAMINANTS



        WHO FOOD ADDITIVES SERIES 40





        Prepared by:
          The forty-ninth meeting of the Joint FAO/WHO Expert
          Committee on Food Additives (JECFA)



        World Health Organization, Geneva 1998



    MALTOGENIC AMYLASE

    First draft prepared by
    M.E.V. Apeldoorn
    and
    Dr G.J.A. Speijers,
    Public Health of the Centre for Substances and Risk Assessment
    National Institute of Public Health and Environmental
    Protection (RIVM), Bilthoven, The Netherlands

        1.  Explanation
        2.  Biological data
            2.1 Biochemical aspects
            2.2 Toxicological studies 
                2.2.1   Acute toxicity studies
                2.2.2   Short-term toxicity studies
                    2.2.2.1 Rats
                2.2.3   Long-term toxicity/carcinogenicity studies
                2.2.4   Reproductive toxicity studies
                2.2.5   Special studies on genotoxicity
                2.2.6   Special study on skin irritation
                    2.2.6.1 Rabbits
                2.2.7   Special study on eye irritation
                    2.2.7.1 Rabbits
                2.2.8   Special study on immune response
                    2.2.8.1 Guinea-pigs
                2.2.9   Other relevant safety data
            2.3 Observations in humans
        3.  Molecular genetic methods used to clone and express the
             Bacillus stearothermophilus maltogenic amylase gene
        4.  Estimates of human consumption
        5.  Comments
        6.  Evaluation
        7.  References

    1.  EXPLANATION

        This enzyme preparation is a maltogenic amylase produced by
    submerged fermentation of a non-pathogenic and non-toxicogenic strain
    of  Bacillus subtilis which, by recombinant DNA techniques, contains
    the  amyM gene from  Bacillus stearothermophilus coding for
    maltogenic amylase.  This maltogenic amylase has not been previously
    evaluated by the Committee.

        Formulations of maltogenic amylase are used in the baking and
    starch industry. It is an exo-acting maltogenic amylase enzyme
    (E.C.3.2.1.133, glucan alpha-1,4-maltohydrolase) and catalyses the
    hydrolysis of alpha-1,4-glucosidic linkages in amylose, amylopectin
    and related glucose polymers. Maltose units are successively removed
    from the non-reducing end of the polymer chain until the molecule is
    degraded or, in the case of amylopectin, a branch-point is reached.
    The Committee noted that the human intake of this recombinant
    maltogenic amylase resulting from its intended use in the baking and

    starch industry would be low and that the material consumed would not
    be the active maltogenic amylase but a heated, denatured material.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

        No information was available.

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

        One study on the acute toxicity of maltogenic amylase has been
    reported as reported in Table 1.



        Table 1.  Acute toxicity studies

                                                                                                    

    Species    Strain    Sex     Route         Duration     LC50            Reference
                                                            (mg/litre)
                                                                                                    

    1Rat       CD        m, f    inhalation    4 h          >1.59           Andersen et al., 1987
                                                                                                    

    1   The test was performed according to OECD Guidelines for Testing of Chemicals. During exposure 
        no mortality or signs of reaction were seen. The highest concentration attainable was 1.59 
        mg/litre. Only a summary is available; the original report was not submitted (Andersen 
        et al., 1987).
    


    2.2.2  Short-term toxicity studies

    2.2.2.1  Rats

        In a 4-week range-finding study performed according to prevailing
    guidelines, CD rats received 0, 0.5, 2.5 and 10% maltogenic amylase in
    their diet. Decreased food intake was seen at 10% and a similar trend
    was also apparent in males given 2.5%. A dose-related reduction in
    body weight gain was seen in males, attaining statistical significance
    in rats receiving 2.5 and 10% (p<0.05 and p<0.01, respectively). A
    slight reduction was also evident in females receiving 10%. The
    reduced food intake and reduced weight gain was associated with
    reduced palatability and nutritional value. Only a summary was
    available; the original report was not submitted (Andersen  et al., 
    1987).

        Groups of 20 male and 20 female CD rats (age 4-6 weeks; bw 105-138
    g for males and 92-123 g for females) received for 13 weeks 0, 5000,
    15 000 or 50 000 mg maltogenic amylase/kg diet (equal to 0, 390, 1200
    or 4000 mg/kg bw per day for males and 0, 440, 1300 or 4300 mg/kg bw
    per day for females). All animals were observed at least twice daily
    (once daily at weekends). Body weight and food consumption were
    determined weekly. Water consumption was assessed by visual
    examination. Ophthalmoscopy was performed in all animals before the
    start of the experiment and in animals of the control and highest dose
    groups at week 12. After week 12 blood was collected (10/sex/group)
    for haematology (Hb, Ht, Er, MCHC, MCV, MCH, Leu, Diff, platelet
    counts) and clinical chemistry (ALAT, ASAT, OCT, GGT, BUN, creatinine,
    glucose, total bilirubin, total protein, electrophoretic protein
    fractions, Na, Cl, Ca, P). After 13 weeks all animals were killed.
    Organ weights (absolute weight and weight relative to body weight) of
    12 organs) were determined. Macroscopy of all animals and microscopy
    of about 30 tissues/organs of animals in the control and highest dose
    groups were carried out.

        No mortality was seen and no clinical signs due to treatment were
    observed. Ophthalmoscopy did not show any abnormalities. A slight
    decrease in food intake of males and females given 50 000 mg/kg was
    seen, accompanied by a significantly decreased body weight gain. The
    overall efficiency of food utilization of males and females in the
    highest dose group was slightly lower than that of controls. The lower
    food intake was associated with increased scattering during the first
    weeks of treatment, suggesting unpalatability of the diet. The weight
    gain of males at 15 000 mg/kg diet was also slightly lower than that
    of controls (<10%, not statistically significant), but food
    consumption was not affected at this dose level. Water consumption was
    not influenced. Haematology did not reveal abnormalities. Clinical
    chemistry showed significantly (p<0.05) lower plasma glucose levels
    in males at the highest dose level. Slightly higher plasma total
    protein levels with associated small increases in the concentrations
    of various globulins, resulting in slightly lower A/G ratios, were
    seen in animals at the highest dose level. Small but significant
    decreases in plasma Cl levels in females given 15 000 or 50 000 mg/kg

    were observed, but inter-group differences were small. The changes
    seen in clinical chemical parameters were not considered to be of
    toxicological significance. Organ weights revealed significantly lower
    absolute and relative thyroid weights in males at 50 000 mg/kg.
    Females revealed a significantly lower absolute lung weight at 50 000
    mg/kg. Macroscopy and microscopy did not reveal any treatment related
    abnormalities. The NOAEL in this study was 15 000 mg/kg maltogenic
    amylase in the diet (equal to 1200 mg/kg bw per day) (West, 1986).

    2.2.3  Long-term toxicity/carcinogenicity studies

        No information was available.

    2.2.4  Reproductive toxicity studies

        No information was available.

    2.2.5  Special studies on genotoxicity

        The results of studies on genotoxicity are described in Table 2.



        Table 2. Studies on the genotoxicity of maltogenic amylase

                                                                                                                                  

    Test system           Test organism              Concentration             Result                          Reference
                                                                                                                                  

    Ames-test1            Salmonella typhimurium     0.1-10 mg/ml without      Negative without and with       Pedersen (1986)
                          TA98, TA100, TA1535,       and with S9               S9.
                          TA1537                                               Toxic at 3.3 and 10 mg/ml 
                                                                               without S9 for TA1537.

    in vitro gene         mouse lymphoma L5178Y      0.158-5 mg/ml (three      No cytotoxicity;                Clare (1990)
    mutation test2        cells                      expts. in dupl)           negative without and with 
                          (HGPRT-locus)                                        S9.

    in vitro              human lymphocytes          1-4 mg/ml                 About 67% mitotic inhibition    Bak (1990)
    chromosomal                                                                at 4 mg/ml without S9. No 
    aberration test3                                                           mitotic inhibition up to 4 
                                                                               mg/ml with S9. No 
                                                                               chromosomal aberrations were 
                                                                               induced.

    in vivo               CD-rats                    2 groups of 15/sex 0      At 5000 mg/kg bw limited        Bootman et al. 
    chromosomal                                      and 5000 mg/kg bw         depression of mitotic index.    (1986)
    aberration test                                  2 groups of 5/sex 200     No induction of chromosomal 
                                                     and 1000 mg/kg bw         abnormalities was seen at 
                                                                               any dose-level.
                                                                                                                                  

    1   Crude enzyme preparations like maltogenic amylase contain free amino acids like histidine. This makes the interpretation
        of results from a standard "plate incorporation assay" difficult. Therefore a modified liquid culture assay was applied.
        Bacteria were exposed to 5 concentrations of maltogenic amylase in a phosphate-buffered nutrient broth for 3 h. After
        incubation, enzyme was removed by centrifugation prior to plating. The test was performed without and with S9. Solvent
        control and positive control assays were included. Toxicity was seen at 3.3 and 10 mg/ml without S9 for TA1537. No
        mutagenic activity of maltogenic amylase was seen in this assay.

    Table 2 (continued)

    2   The test was performed according to OECD Guideline No 476. Three independent experiments were carried out in duplicate.
        Exposure time was 2 h in the absence as well as the presence of S9. No cytotoxicity was seen up to 5 mg/ml without and
        with S9. In experiment 1 concentrations of 0, 158, 0.5, 1.58 and 5 g/ml were used and in experiments 2 and 3
        concentrations of 1, 2, 3, 4 and 5 mg/ml were used. Without S9 no increases of mutation frequencies were seen in
        experiment 1. A positive effect was seen in experiment 2 at 5 mg/ml with a dose relationship confirmed by linear
        regression analysis. This effect was not reproducible in the 3rd experiment. With S9 no increases of mutation
        frequencies were seen in experiment 1. In experiment 2 a positive effect was seen at the second lowest dose of 2 mg/ml
        and in experiment 3 at the three lowest doses of 1, 2 and 3 mg/ml, while at the higher dose levels with S9 in experiment
        2 and 3 no positive effects were seen. In conclusion no reproducible dose-related increases in mutation frequencies were
        observed.
    3   The test was performed according to OECD Guideline No. 473. In a preliminary cytotoxicity study mitotic inhibition was
        seen at 4 mg/ml without and with S9. Concentrations of 1, 2 and 4 mg/ml were used in the main study. The test was
        performed without and with S9. Solvent control and positive control assays were included. Human lymphocytes were
        incubated for 24 hours in the absence of S9 and for 2 h in the presence of S9. Cells were harvested 24 h after the start
        of the experiment. Without S9 about 67% mitotic inhibition was seen at 4 mg/ml. With S9 no mitotic inhibition was seen.
        No induction of aberrant metaphases was seen.
    4   In a preliminary cytotoxicity study with doses of 40-5000 mg maltogenic amylase/kg bw in distilled water (no untreated
        group was included) a slight reduction of mitotic index was seen at 5000 mg/kg bw after 24 hours (mean mitotic indices
        5.2, 6.0, 7.1 and 4.3 at 40, 200, 1000 and 5000 mg/kg bw, respectively). Therefore 5000 mg/kg bw was selected as the
        highest dose in the main study. Groups of 15 male and 15 female CD rats received a single oral dose by gavage of 0 or
        5 mg/kg bw in distilled water and 5 male and 5 female rats/group were killed 6, 24 and 48 hours after administration.
        Groups of 5 male and 5 female rats received a single oral dose by gavage of 200 or 1000 mg/kg bw in distilled water.
        These animals were killed 24 hours after administration. A positive control group was included. No significant increases
        in aberrant cells were found at any of the concentrations used. At 5000 mg/kg bw a not statistically significant, but
        biologically significant, reduction of the mitotic index was seen in female animals, which may be an indication of
        exposure of target cells. In males at 5000 mg/kg bw the reduction of the mitotic index was smaller that in females.
    


    2.2.6  Special study on skin irritation

    2.2.6.1  Rabbits

        In a 4-hour skin irritation study in New Zealand White rabbits
    performed according to OECD Guidelines, a primary skin irritation
    score of 0.21 was found. Maltogenic amylase was classified as
    "non-irritant" for the skin. Only a summary was available; the
    original report was not submitted (Andersen  et al., 1987).

    2.2.7  Special study on eye irritation

    2.2.7.1  Rabbits

        In an eye-irritation study with New Zealand white rabbits
    performed according to Buehler (1965), maltogenic amylase was found to
    be "non-irritant" as only negligible reactions were seen (Andersen 
     et al., 1987). Only a summary was available; the original report was
    not submitted.

    2.2.8  Special study on immune response

    2.2.8.1  Guinea-pigs

        In a delayed contact hypersensitivity study with Dunkin-Hartley
    guinea-pigs, maltogenic amylase did not display positive reactions
    after a challenge application, while all animals exposed to
    dinitrochlorobenzene showed positive reactions. It was concluded that
    maltogenic amylase did not cause skin sensitization. Only a summary
    was available; the original report was not submitted (Andersen 
     et al., 1987).

    2.2.9  Other relevant safety data

        Maltogenic amylase did not reveal antibacterial activity in an
    assay performed according to the method recommended by the Committee
    (Annex 1, reference 58, Annex I).Only a summary was available;
    original report not submitted (Andersen  et al., 1987).

    2.3  Observations in humans

        No information was available.

    3.  MOLECULAR GENETIC METHODS USED TO CLONE AND EXPRESS THE 
    B. STEAROTHERMOPHILUS MALTOGENIC AMYLASE GENE

        The maltogenic amylase gene  (amyM) was derived from the
    spore-forming bacterium  Bacillus stearothermophilus. As a first
    step, partially digested  (MboI) chromosomal DNA of 
     B. stearothermophilus was cloned into pACYC184 and was transfected
    into  E. coli K12 strain 802 (a non-pathogenic  E. coli strain). A
    transformant containing a14 kb plasmid (pDN400) was selected due to
    its amylase expression (approx. 10 kb  B. stearothermophilus 

    chromosomal DNA was present in pACYC184). PDN400 was partially
    digested with  Sau3A and DNA fragments were cloned into pBD64 (a 
     B. subtilis replicating vector and transformed into  B. subtilis 
    168 strain DN314 (strain 304 harbouring plasmid PC1B110)). One 
     B. subtilis clone with a  CAMR and  Amy+phenotype was selected;
    plasmids were prepared and transformed into  B. subtilis 304. After
    this re-transformation, all clones contained a plasmid pDN452,
    harbouring 2.8 kb of  B. stearothermophilus chromosomal DNA.
    Thereafter, a spontaneous mutant with increased genetic stability and
    displaying an increased amylase yield was selected (i.e. DN520,
    containing plasmid pDN520). However, the selection procedure was not
    described. pDN520 appeared to have a mutation  (cop-520) in the
    replication origin, giving the plasmid a higher segregational
    stability. The  B. stearothermophilus chromosomal DNA insert of
    pDN520 was transferred to pUB110, giving rise to pDN623, which was
    transfected into  B. subtilis host DN623. From a final selection step
    (selection procedure was not given) strain DN1413 was obtained; this
     B. subtilis strain had lost its sporulation phenotype.  B. 
     subtilis DN1413 was further used as the actual producer strain
    having the following genotype: pDN1413;  KanR,  Amy+,  cop-520).
    During the adaptation for growth on industrial media DN1413 lost the
    methioanine requirements of DN623.

        pDN1413 was thoroughly characterized (mostly by sequence analysis)
    and both the  amyM gene and its strong promoter were identified.
    pDN1413 was derived exclusively from DNA from 110 and chromosomal DNA
    of the donor microorganism. pDN1413 appeared to be integrated into the
    genome of the producer strain DN1413, which gave it extreme genetic
    stability. Reversion to a sporulating phenotype occurred at a very low
    frequency (1:108).

        DN1413 overproduces maltogenic amylase (as confirmed by
    immunological methods), which has an apparent relative molecular mass
    of 75 000 (as determined by SDS page).

        Both the recipient  (B. subtilis) and the donor strain 
     (B. stearothermophilus) are non-pathogenic to humans and animals
    (Diderichsen & Christiansen, 1988; Anonymous, 1991, 1996; de Boer &
    Diderichsen, 1991; Toft, 1996).

        Although the plasmid pDN1413 carries the kanamycin resistance
    gene, it is unlikely that the gene can be transferred, since it is
    stably integrated into the host genome and no plasmid DNA could be
    detected in the end product (0.1 ng plasmid in 1 g enzyme). On
    theoretical grounds (i.e. almost the entire DNA sequence of pDN 1413
    was determined) it can be concluded that Shiga-like toxins will not be
    produced and will therefore be absent from the final product.

    4.  ESTIMATES OF HUMAN CONSUMPTION

        "Worst case" residues were calculated assuming that the product is
    retained in bread and syrup (Toft, 1996).

     a) Bread

        The preparation of maltogenic amylase Novamyl 1500 MG for use in
    baking has an activity of 1500 MANU1/g (corresponding to a content
    of 6% total organic substance (TOS)). The maximum recommended dosage
    is 60 g per 100 kg flour. Using a standard recipe, 100 kg flour would
    result in 140 kg bread, giving a theoretical content of 429 mg Novamyl
    1500 MG/kg bread and related products. Based on a daily intake of 158
    g bread, the daily intake per person of maltogenic amylase would be 68
    mg, corresponding to 1.13 mg/kg bw per day for an average person
    weighing 60 kg (corresponding to 0.068 mg TOS/kg bw).

     b) Sugar

        The preparation of maltogenic amylase Maltogenase 4000 L for use
    in the starch industry has an activity of 4000 MANU/g (corresponding
    to a content of 16% TOS). The maximum recommended dosage is 5 g/kg
    starch. Saccharification of 1 kg starch results in 1 kg sugar, giving
    a theoretical content of 5 g Maltogenase 4000 L/kg sugar. Based on a
    daily intake of 143 g sugar and high fructose corn syrup obtained from
    a surveillance in the USA, the daily intake per person of maltogenic
    amylase would be 715 mg, corresponding to 11.9 mg/kg bw per day for an
    average person weighing 60 kg (corresponding to 1.9 mg TOS/kg bw).

    5.  COMMENTS

        Available data that were reviewed included the genetic
    modification procedures employed for the production of the enzyme
    characterization of the producing organisms, the fermentation process,
    acute and short-term toxicity studies in animals, and genotoxicity
    studies.

        The Committee noted that well-documented non-pathogenic and
    non-toxicogenic strains of microorganisms  (Bacillus subtilis, 
     Escherichia coli K12 and  Bacillus stearothermophilus) had been
    used in the genetic modification procedures. The final vector used
    (pUB110) is well characterized and has been used for several years as
    a cloning vehicle for  Bacillus subtilis. The plasmid construct
    pDN1413, containing the  amyM gene, was introduced into  B. 
     subtilis  (a derivative of strain 168) using standard transformation
    procedures. Although the plasmid pDN1413 carries the kanamycin
    resistance gene, it is not likely that the gene can be transferred,
    since it is stably integrated into the host genome and no plasmid DNA
    could be detected in the end product (sensitivity of analytical
    method:  0.1 ng of plasmid in 1 gram of enzyme). The entire DNA
    sequence of pDN 1413 was determined, which confirmed that Shiga-like
    toxins will not be produced.

                   

    1 One MANU is the amount of enzyme required to cleave 1 micromole of
    maltriose per min under standard conditions.

         B. subtilis was grown under properly controlled conditions in
    media containing ingredients commonly used in the production of
    food-grade substances by fermentation.

        From the evaluation of the recombinant DNA procedures being
    employed, the Committee concluded that the final construct should be
    regarded as a safe source of maltogenic amylase.

        The product tested in the toxicological studies was a concentrated
    material (enzyme activity 35900 maltogenic amylase units/g). It was
    produced according to the standard production process except that the
    formulation/standardization was omitted and the product was
    lyophilized.

        In a 90-day dietary study in rats the lyophilized test compound
    caused significantly decreased body-weight gain accompanied by a
    slight decrease in food consumption in males and females at the
    highest dose-level of 5% of the diet. At the same level significantly
    lower thyroid weights were seen in males as well as females. At the
    next lower dose-level of 1.5% of the diet no statistically significant
    treatment-related findings were observed. The NOEL in this study was
    1.5% of the diet, equal to 1200 mg/kg bw per day.

        The test compound had no effects in  in vitro gene mutation
    studies in bacteria and mammalian cells and chromosomal aberration
    tests  in vivo and  in vitro were consistently negative.

        The test compound did not cause skin or eye irritation in rabbits
    and did not produce skin sensitization in a delayed contact
    hypersensitivity assay in guinea-pigs.

    6.  EVALUATION

        The Committee allocated a temporary ADI "not specified" to
    maltogenic amylase derived from this recombinant strain pending
    deletion of the "tentative" qualification of the specifications.

    7.  REFERENCES

    Andersen, J.R, Diderichsen, B.K., Hjortkjaer, R.K., De Boer, A.S.,
    Bootman, J., West, H., & Ashby, R. (1987) Determining the safety of
    maltogenic amylase produced by r-DNA technology.  J. Food Prot., 
    50(6): 521-526.

    Anonymous (1991) Plasmid DNA in product. Confidential data from Novo
    Nordisk (Ref.: F/912211/BBJphi) (Submitted to WHO by Novo Nordisk A/S,
    Bagsvaerd, Denmark).

    Anonymous (1996) Immuno identification between maltogenic amylase from
    donor organism and cloned product. Confidential data from Novo Nordisk
    (Ref.: AT/96-10-21) (Submitted to WHO by Novo Nordisk A/S, Bagsvaerd,
    Denmark).

    Bak, J. (1990)  In vitro assessment of the clastogenic activity of
    Ts-25-amylase in cultured human lymphocytes. Unpublished study No.
    9003 (File: F-902403) from Novo Nordisk A/S, Enzyme Toxicology
    Laboratory, Bagsvaerd, Denmark (Submitted to WHO by Novo Nordisk A/S,
    Bagsvaerd, Denmark).

    Bootman, J., Hodson-Walker, G., & Dance, C. (1986) SP-295:
    Investigation of effects on bone-marrow chromosomes of the rat after
    acute oral administration. Unpublished report No. 85/NLE008/735 from
    Life Science Research Ltd, Suffolk, England (Submitted to WHO by Novo
    Nordisk A/S, Bagsvaerd, Denmark).

    Buehler, E.V. (1965) Delayed contact hypersensitivity in the
    guinea-pig.  Arch. Dermatol., 91, 171.

    Clare, C.B. (1990) Study to determine the ability of maltogenic
    amylase to induce mutations to 6-thioguanine resistance in mouse
    lymphoma L5178Y cells using a fluctuation assay. Unpublished report
    No. 2MLRENOD.021 from Hazleton Microtest, Heslington, York, England
    (Submitted to WHO by Novo Nordisk A/S, Bagsvaerd, Denmark).

    de Boer, A.S. & Diderichsen, B. (1991) On the safety of  Bacillus 
     subtilis and  B. amyloliquefaciens: a review.  Appl. Microbiol. 
     Biotechnol., 36: 1-4.

    Diderichsen, B. & Christiansen, L. (1988) Cloning of a maltogenic
    alpha-amylase from  Bacillus stearothermophilus. FEMS Microbiol. 
     Lett., 56, 53-60.

    Pedersen, P.B. (1986) SP 295 (Batch No. PPY 1670): Testing for
    mutagenic activity with  Salmonella typhimurium TA 1535, TA 1537, TA
    98 and TA 100 in a liquid culture assay.Unpublished study No. E.0285
    from Novo Nordisk A/S Enzyme Microbiological R & D (Submitted to WHO
    by Novo Nordisk A/S, Bagsvaerd, Denmark).

    Toft, A. (1996) Maltogenic amylase (Novamyl, Maltogenase): Data
    relevant to the toxicological evaluation of the maltogenic amylase
    expressed in  Bacillus subtilis carrying the gene coding for
    maltogenic amylase from  Bacillus stearothermophilus inserted by
    r-DNA techniques. Confidential report No. QR-9616659 AT/JetB from Novo
    Nordisk Enzyme Regulatory affairs (Submitted to WHO by Novo Nordisk
    A/S, Bagsvaerd, Denmark).

    West, H.A. (1986) SP 295: Toxicity study by dietary administration to
    CD rats for 13 weeks. Final Report. Unpublished report No.
    85/NLE006/742 from Life Science Research Ltd. Suffolk, England
    (Submitted to WHO by Novo Nordisk A/S, Bagsvaerd, Denmark).

    


    See Also:
       Toxicological Abbreviations